The present invention concerns an apparatus and method for changing the solvent environment of a solution containing macromolecules. The apparatus and method are useful in a variety of preparative and analytical procedures involving macromolecules in solution or in suspension, and suspended macromolecular aggregates. The necessity for such a method arises whenever it is desired to change the solvent composition, pH or ionic strength, or whenever it is desired to separate the macromolecules in solution or suspension from low molecular weight substances.
A variety of methods for exchanging one solvent or suspending medium for another are well known to those skilled in the art. With the exception of specific methods exploiting the chemical properties of specific macromolecular species, those methods generally applicable to macromolecular solutions and suspensions all exploit the difference in size between the macromolecules and the molecules of the solvent. Most commonly, a solvent or suspension medium is composed of one or more substances having molecular weights less than 1000 while the macromolecules are generally substances having molecular weights greater than 1000, although the distinction is arbitrary. The important factors are not the absolute molecular weights, but the differences in molecular weight and molecular size between the molecules of the medium and the macromolecules. For example, most of the described prior art methods, and the method of the present invention could, in principle, be used to separate two macromolecular species from each other, provided there was a sufficiently large molecular size difference between the two species.
Dialysis is the name given to processes based upon diffusion of molecules through a semi-permeable membrane. A membrane is chosen which is permeable to the small molecules of the medium but impermeable to the macromolecules. The exchange of molecules of the original medium, medium A, with molecules of the desired new medium, medium B, through the membrane, occurs by diffusion. Consequently, the process is slow. At equilibrium, the fraction of medium A remaining with the macromolecular sample will equal the relative volumes of the macromolecular sample and the volume of medium B to which it is exposed. For example, if one volume of macromolecule sample containing medium A is dialyzed against nine volumes of medium B, the concentration of medium A in the sample will be reduced to not less than one-tenth, under ideal conditions, at equilibrium. Alternatively, the macromolecular sample may be dialyzed against several changes of smaller volumes of medium B, to achieve the desired result. The process consumes large amounts of medium B, which can only be partially conserved at the cost of greatly increasing the number of manipulative steps. Additional limitations on the dialysis method include the fact that the method does not work well where there are interactions between medium A and medium B such as the formation of a precipitate, which could interfere with the process. In addition, certain substances interact with the dialysis membranes commonly used, such that they fail to diffuse freely through the membrane.
Gel filtration employs granules of cross-linked polymer having a porous or spongy structure. The structure of the granules is controlled such that molecules of the medium can diffuse within the granules but molecules greater than a certain size limit are excluded. In a typical gel filtration process, a column of the polymer beads is constructed, equilibrated with medium B, and the macromolecular sample in medium A is applied to the column and eluted with medium B. Since the molecules of medium A are able to penetrate the interior of the polymer granules, they tend to travel more slowly through the column than do the macromolecules, as the column is eluted. The macromolecules are confined to the spaces between the granules, and tend to migrate more rapidly, since they follow a more direct path. Consequently, as the column is eluted, solvent A is impeded and the macromolecules exit the column in medium B. One disadvantage of the procedure is that it generally introduces some dilution of the macromolecules. In addition, the procedure is time consuming and requires complex chromatography equipment, including fraction collecting equipment and means for assaying the peak fractions containing the macromolecules.
Other chromatographic methods have been employed to effect changes of medium. Macromolecules applied to the column in medium A are eluted with medium B. Such methods are applicable only under certain circumstances. The desired macromolecule must be adsorbed or otherwise retained on the column in medium A and released from the column by medium B. The proper conditions must be determined empirically and are not usually applicable from one macromolecular species to the next. The above-cited disadvantages for gel filtration chromatography are applicable to other chromatographic methods, namely dilution of the sample and need for expensive equipment. In addition, chromatographic methods are often time consuming.
Various specific methods exploiting properties of specific macromolecules are also known. For example, nucleic acids may be precipitated from medium A by the addition of ethanol, and the precipitate may then be redissolved in medium B. Such methods, by their very nature, are only useful in specialized circumstances, and care must be taken to insure that biological activity is retained if desired and that recovery of the macromolecules is quantitative.
Membrane filtration is a term applied to a variety of methods using a selectively permeable membrane in combination with a pressure differential across the membrane. All of the molecules which can pass through the membrane will tend to travel from the high pressure side to the low pressure side of the membrane. For example, a solution containing macromolecules can be concentrated with respect to its macromolecule content by the removal of solvent molecules through a membrane filter such that the fluid volume is reduced while the total mass of macromolecules remains the same. An exchange of media can be effected by employing a concentration step for the removal of solvent A, followed by redilution with solvent B. As a practical matter, the concentration factor of a single step is limited, and complete exchange of media requires several cycles of concentration and redilution. The limitation on concentration is due in part to the accumulation of concentrated macromolecules at the membrane surface resulting in the formation of a macromolecular gel which restricts further flow of material through the membrane. The phenomenon is referred to as concentration polarization and may be counteracted to some extent by the application of stirring at the surface of the membrane. Concentration polarization can also be reduced by causing flow of the solution in a direction parallel or tangential to the membrane surface.
Apparatus for producing tangential flow typically consists of convoluted tubular or lamellar structures through which the solution to be concentrated is required to flow. For a description of concentration polarization and methods for reducing it, see Millipore Product Information Bulletin, "Molecular Filtration", Cat. No. LAR 8010/P, Millipore Corp., Bedford, Mass. 01730. An apparatus exploiting the principle of tangential flow during membrane filtration is disclosed in U.S. Pat. No. 3,591,493, issued July 6, 1971 to Zeineh, and U.S. Pat. No. 3,820,661, issued June 28, 1974 to Pages. In order to effect a replacement of medium A with medium B in such an apparatus, it would be necessary for the macromolecule solution to leave its original container, pass through a series of tubes into the concentrating apparatus and then be rinsed out of the concentrating apparatus into a new container. The replacement of medium would require two steps, first a concentration step, then a dilution step with medium B. The necessity of transferring the sample from its original container and conducting it through the concentrating apparatus with concomitant exposure to a large surface area of membrane filter necessarily reduces the efficiency of sample recovery and limits the method to samples sufficiently large to fill the apparatus. In addition, apparatus of this type is often expensive to buy and difficult to maintain.
Immersible filters have been employed for a variety of purposes, using a variety of designs in the prior art. Immersible filters generally are constructed with the filter element enclosing an interior space. The interior communicates with the filtrate reservoir by means of a tube which may also function as a passageway for backwashing the filter, in some embodiments. U.S. Pat. No. 195,423, issued Sept. 18, 1877 to Vent, discloses a cylindrical or spherical immersible filter having an internal space which may be connected to vacuum. The patent further discloses a filter conforming approximately to the shape of the vessel containing it, in order to provide maximum filter surface area relative to the volume of fluid to be filtered. The filter shape is chosen so as to provide a structure which may be oscillated around pivot points connected to oscillating means outside the container.
The use of an immersible filter in order to maintain a constant volume in the vessel containing the liquid to be filtered is disclosed in U.S. Pat. No. 1,043,455, issued Nov. 5, 1912 to Neil. Suction is applied to the interior of the filter to withdraw liquid from the reservoir through the filter and means are provided for introducing additional liquid to be filtered at approximately the same rate.
U.S. Pat. No. 1,435,972, issued Nov. 21, 1922 to Olsson, discloses the use of vacuum filtration with an immersed filter element, for use in a process of purifying oil.
U.S. Pat. No. 3,630,360, issued Dec. 28, 1971 to Davis, et al. discloses a method of filtering fine particles from liquids by applying a slight vacuum to the interior of an immersed filter thereby drawing off the liquid. The filter element is inverted to reduce filter clogging by gravitational settling of the particles, and to facilitate backwashing.